Previous work in our laboratory and elsewhere has established that intracellular redox status measured in terms of intracellular glutathione (GSH) levels regulates T cell function. Inflammatory responses such as the activation of NF-kappaB are up-regulated under oxidative stress (when GSH is depleted) whereas activation events required for T cell (TCR) signaling, including tyrosine kinase phosphorylations and calcium influx, are down-regulated under these conditions but favored when intracellular GSH levels are normal. Consistent with these findings, intracellular GSH tends to be depleted in a wide variety of diseases (e.g., HIV infection, sepsis, diabetes, neoplasia) in which T cell function is often impaired. To define the biochemical mechanisms that underlie the redox regulation of TCR signaling, we propose here to combine the 11-color Fluorescent Activated Cell Sorter (Hi-D FACS) technology that we have developed to measure intracellular redox status in human T cell subsets with Hi-D FACS methods to quantitate phosphorylation of individual kinases in these T cell subsets. By examining the functional consequences of altering redox status (depleting/repleting GSH) in freshly isolated T cells from healthy donors, and by probing the relationship between redox status and T cell function in patients with various diseases, we contribute significantly to basic and clinical understanding of the impact of GSH depletion on immune function. Further, since treatment with non-toxic cysteine pro-drugs can replenish GSH, our findings may open the way to introduction of adjunct therapies to increase immune surveillance and bolster responses to infection. Finally, the technology we develop here will provide new and better ways to monitor disease progress and evaluate therapeutic interventions.